Mitochondria are a major target for environmental and endogenous toxic chemical species and participate in pathways that lead to cellular oxidative stress. First, the inner membrane of these organelles contains the electron transport chain, where reactive oxygen species, such as superoxide anions and hydrogen peroxide, are produced as side reactions of normal aerobic respiration. These molecules cause damage to cellular components such as lipids, proteins and DNA that can lead to genetic mutations, permanent alteration of cell function and ultimately disease. Second, mitochondria contain their own DNA genome (mtDNA) that is itself particularly susceptible to damage and mutation. Compared to the nucleus, there is a paucity of fundamental information regarding the pathways and mechanisms that allow mtDNA to resist oxidative, as well as other types of, DNA damage. The investigators preliminary results using a yeast model system revealed that multiple pathways are at work in mitochondria to resist oxidative DNA damage and mutagenesis. Importantly, these pathways appear to be largely recombination-independent, and therefore hold significance for similar systems operating in humans, where mtDNA recombination is less prevalent. The overall goal of this project is to further dissect these oxidative DNA damageresistance pathways that protect and maintain the mitochondrial genome using a yeast model system and a combination of genetic, cytological and biochemical strategies. There are three specific aims of the proposed research project: Specific Aim1 is to examine the role of the abundant mitochondrial DNA-binding protein, Abf2p, in mtDNA damage resistance, by testing the hypotheses that it is involved directly in preventing DNA damage by acting as a shielding agent or is involved in facilitating the initial steps of mitochondrial base-excision repair; Specific Aim 2 is to examine the role of the mitochondrial DNA helicase, Pif1p, in mtDNA damage resistance, by testing the hypotheses that it is involved in regulating the rate or timing of mtDNA replication (a DNA damage checkpoint-like function) or is involved in facilitating the initial steps of mitochondrial base-excision repair; Specific Aim 3 is to complete the analysis of nine mutant yeast strains that exhibit a synthetic-petite phenotype with NTG1 null mutations, which will identify additional factors that define or influence mtDNA damage-resistance pathways in mitochondria. Together with the other projects in this Program, completion of these aims will not only elucidate strategies used to resist mtDNA damage, but also how various pathways cooperate to provide overall cellular DNA damage resistance.